HIV is a retrovirus that causes immunosuppression in humans (HIV disease), which culminates in a disease complex known as the acquired immunodeficiency syndrome (AIDS). This retrovirus is a member of the lentivirus subfamily, which includes non-oncogenic retroviruses that cause persistent (chronic active) infections in diseases with long incubation periods. These viruses usually infect cells of the immune system (particularly macrophages and T cells) and cause cytopathic effects in infected cells, such as syncytia and cell death. Lentiviral infections are not cleared by the immune system, and lead to accumulated immunologic damage over a period of many years.
The treatment of HIV disease has been significantly advanced by the recognition that combining different drugs with specific activities against different biochemical functions of the virus (combination therapy) can help reduce the rapid development of drug resistant viruses that was seen in response to single drug treatments. However, even with combination therapies, multi-drug resistant strains of the virus have emerged. There is therefore a continuing need for the development of new anti-retroviral drugs that act specifically at different steps of the viral infection and replication cycle.
The viral infectivity factor (Vif; also referred to as Sor or Q) encoded by HIV-1 is a small basic Mr˜23,000 phosphoprotein that is synthesized in a Rev-dependent manner during the late stages of virion production. Homologs of Vif exist in all lentiviruses, with the only exception being equine infectious anemia virus (EIAV) (Oberste & Gonda, Virus Genes 6; 95–102, 1992). There is significant conservation among vif open reading frames (ORFs) of the different lentiviruses (Sonigo et al., Cell 42; 369–382, 1985).
Although Vif has no effect on the release of HIV particles from infected cells, it enhances their infectivity fifty to one-hundred fold, in a manner that depends on the producer cells, and is independent of the target cells used to assay the infectivity. It is necessary for HIV-1 replication in vivo and in nonpermissive cells, which include T lymphocytes and macrophages and several leukemic T cell lines, but it is irrelevant in many other cells termed permissive (Gabuzda et al., J. Virol. 66, 6489–6495, 1992; Madani & Kabat, J. Virol. 72, 10251–10255, 1998; Simon et al., Nat. Med. 4, 1397–1400, 1998; Sheehy et al., Nature 418, 646–650, 2002). Consequently, HIV-1(Δvif) that has a deletion or mutation in its vif gene can efficiently replicate in permissive cell lines. Furthermore, the resulting HIV-1(Δvif) virions can also infect nonpermissive cells, resulting in proviral DNA integration and in production of virus-encoded proteins that are packaged with viral RNA into progeny virions that appear to have a normal composition (Gaddis et al., J. Virol. 77, 5810–5820, 2003; Ochsenbauer et al., Gen. Virol. 78, 627–635, 1997). However, these HIV-1(Δvif) virions that are derived from nonpermissive cells have been imprinted in a manner that severely inhibits reverse transcription during the subsequent cycle of infection (von Schwedler et al., J. Virol. 67, 4945–4955, 1993; Courcoul et al., J. Virol. 69, 2068–2074, 1995; Simon & Malim, J. Virol. 70, 5297–4305, 1996; Dettenhofer et al., J. Virol. 74, 8938–8945, 2000; Goncalves et al., J. Virol. 70, 8701–8709, 1996). Because these virions are inactive in all target cells including permissive or nonpermissive cells that contain Vif, the imprinting may be irreversible (Gabuzda et al., J. Virol. 66, 6489–6495, 1992; von Schwedler et al., J. Virol. 67, 4945–4955, 1993; Courcoul et al., J. Virol. 69, 2068–2074, 1995). This imprinting phenomenon, which is diagrammed in FIG. 1, has made elucidation of Vif function difficult because it is imposed in the cells producing virions but its outcome only becomes evident in the subsequently infected target cells.
The observed cellular specificity is believed to occur because nonpermissive (NP) cells but not permissive (P) cells contain an inhibitor of HIV-1 infectivity, and because Vif counteracts and neutralizes this inhibitor (Madani and Kabat, J. Virol. 27:10251–10255, 1998). Consequently, the vif gene is irrelevant for lentivirus replication in P cells because these cells lack the antiviral inhibitory factor. However, in NP cells the vif-deleted virions become inactivated during their release from the cells. Thus, NP cells can be infected with vif-deleted lentivirus particles that were made in P cells, but these infected NP cells release only noninfectious vif-deleted virions that cannot replicate within a culture or infected animal.
The nonpermissive phenotype is dominant in permissive x nonpermissive heterokaryons (Madani & Kabat, J. Virol. 72, 10251–10255, 1998; Simon et al., Nat. Med. 4, 1397–1400, 1998). This finding suggested that nonpermissive cells have a potent antiviral defense system that would efficiently inactivate HIV-1, were it not neutralized by Vif. This evidence implies that the antiviral inhibitory factor in NP cells can be countermanded and neutralized by Vif. This conclusion is supported by the finding that Vif functions in a species-restricted manner (Simon et al., Embo J., 17:1259–1267, 1998). See also the recent paper by Mariani et al. (Cell 114:21–31, 2003). For example, Vif of African green monkey (SIVagm) does not have an effect in human cells, but it can neutralize the inhibitory factor in African green monkeys. Conversely, SIVagm viruses can replicate in human NP cells engineered to express the HIV-1 vif gene. The HIV Vif protein is therefore believed to neutralize an antiviral factor in nonpermissive human cells, but the HIV Vif protein would be unable to counteract the homologous antiviral factors of AGMs or of more distantly related species, such as mice.
Recently, Sheehy et al. (Nature 418, 646–650, 2002) proposed APOBEC3G (previously referred to as CEM-15), a member of the cytidine deaminase family of nucleic acid editing enzymes (Teng et al., Science 260, 1816–1819, 1993; Harris et al., Mol. Cell. 10, 1247–1253, 2002), as the specific antiviral factor in nonpermissive cells. Most significantly, they reported that expression of APOBEC3G in permissive cell lines converted them to nonpermissive (Sheehy et al., Nature 418, 646–650, 2002).
Additionally, they reported that APOBEC3G is incorporated into HIV-1 virions regardless of whether Vif is present or absent in the producer cells. Since Vif is incorporated in small amounts into HIV-1 virions (Gaddis et al., J. Virol. 77, 5810–5820, 2003; Liu et al., J. Virol. 69, 7630–7638, 1995; Khan et al., J. Virol. 75, 7252–7265, 2001), and since it binds to RNA (Dettenhofer et al., J. Virol. 74, 8938–8945, 2000; Khan et al., J. Virol. 75, 7252–7265, 2001), Sheehy et al. suggested that Vif might bind to the HIV-1 genomic RNA and shield it from inactivation by APOBEC3G in the producer cells and/or in the released virions (Nature 418, 646–650, 2002; Gaddis et al., J. Virol. 77, 5810–5820, 2003), thus acting on the target of APOBEC3G rather than directly on the antiviral protein. Recently, it was shown that APOBEC3G causes cytidine deamination of HIV-1 negative strand DNA during the process of reverse transcription (Lecossier et al., Science 300, 1112, 2003; Zhang et al., Nature 424, 94–98, 2003; Mangeat et al., Nature 424, 99–103, 2003; Harris et al., Cell 113, 803–809, 2003).
It would be particularly advantageous to identify the mechanism by which Vif functions to neutralize APOBEC3G and related factors, because this mechanism (and its biochemical consequences and the pathways that influence this mechanism) would provide important targets in the treatment of HIV disease. Identification of the Vif mechanism would also enable the development of screening assays to test drugs that affect the intracellular expression or the mechanism by which Vif neutralizes the antiviral factor. Such drugs would be expected to interfere with the Vif mediated viral defense, and therefore would be useful to inhibit or interfere with lentiviral infection and/or replication.